Phosphocholinate cardenolides

ABSTRACT

Disclosed herein are cardenolides and related compounds covalently linked to phosphocholine moieties and pharmaceutical formulations comprising such compounds. Also disclosed herein are methods for treating hypertension, premenstrual syndrome (PMS), preeclampsia and polycystic kidney disease using the compounds.

This application is a 371 of PCT/US97/10188 filed May 28, 1997 and alsoclaims the benefit of U.S. Provisional Ser. No. 60/018,458 filed May 28,1996.

FIELD OF THE INVENTION

This invention is related to cardenolides and related compoundscovalently linked to phosphocholine moieties, pharmaceuticalformulations comprising such compounds and their therapeutic anddiagnostic use.

BACKGROUND OF THE INVENTION

Digoxin, digitalis and related cardiotonic agents act on the heart in avariety of ways resulting in an increase in the force of contraction (apositive inotropic effect), a slowing in the rate of atrioventricularconduction thereby leading to improved hemodynamics and renal function.Such agents are useful in treating congestive heart disease and atrialfibrillation.

Congestive heart disease is characterized by an incomplete emptying ofblood from the heart during ventricular contraction, which leads to anenlargement of the heart. When congestive heart disease is treated withcardiac glycosides there is a reduction of heart rate, a more completefilling of the ventricules, and the size of the heart decreases andbegins to return to normal. Atrial fibrillation is a condition in whichthe atria contract much more often then the ventricules, causing thelower heart chambers to be bombarded by impulses. The ventriculesrespond by weakly and inefficiently contracting. When used to treatatrial fibrillation, cardiac glycosides depress the conduction rate,slowing the rate of ventricular contraction and reestablishing asynchronous and effective heart beat.

One of the drawbacks of digoxin and related cardiotonic agents is theirlow therapeutic index. There is a narrow window of concentrations inwhich the drugs are effective for the treatment of congestive heartfailure or atrial fibrillation. In addition, there are numerous adverseside effects resulting from therapy with digoxin and related compounds.These have been extensively reviewed by T. B. Smith et al. in Prog.Cardivas. Dis. 26:21-56, 1984. Side effects of digoxin therapy includeimpotence, weakness and depression. A further drawback is that somepatients do not respond to digoxin treatment. Furthermore, digoxin anddigitalis are only effective to depress blood pressure in patients thathave previously suffered congestive heart failure and are ineffective innormal hypertensive patients.

Hypertension is a common condition and is characterized by elevations ofatrial pressure which could result in secondary organ damage and areduced life span. The condition is currently treated using four generalclasses of drugs: diuretics, anti-adrenergic agents, vasodialtors andangiotensin blockers (Harrison's Principles of Internal Medicine, K. J.Issel Bacher et al., eds., pp. 1172-1176, McGraw Hill, New York, 1980).Each of these agents produce side effects and their use is limited.Cardiac glycosides, used to treat congestive heart disease, areessentially ineffective in treating hypertension.

Numerous investigators have reported the existence of endogenousdigoxin-like factors isolated from various biological fluids. Forexample, Tymiak, A. K. et al. (Proc. Nat. Acad. Sci. USA 90:8189-8193,1993) disclosed a ouabain-like agent isolated from bovine hypothalamus.Goto, A. et Clin. Chem 36:161-162, 1992) also reported finding aouabain-like factor in human urine and bovine plasma. Shaikh, I. M. etal. (J. Biol. Chem. 266:13972-13978, 1991) disclosed a digoxin-likeimmunoreactive factor isolated from mammalian adrenal cortex. The factorwas said to have a molecular mass of 780 daltons comprised of a 390dalton aglycone component plus several sugar moieties.

U.S. Pat. Nos. 5,028,438 and 5,122,371 issued Jul. 2, 1991 and Jun. 6,1992, respectively, describe a biologically active agent havinghypotensive activity in mammals. The agent was said to be derived fromType 1 human female breast cyst fluid and to be immunoreactive withdigoxin antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing a proposed pathway for the synthesis of andthe inter-relationship between the compounds of the present invention.

FIG. 2 is the mass spectral data obtained by HPLC/Atmospheric PressureChemical Ionization Mass Spectroscopy (HPLC/APCI-MS) on AM-496.

FIG. 3 is the mass spectral data obtained by HPLC/APCI-MS on AM-520.

FIG. 4 is the mass spectral data obtained by HPLC/APCI-MS on AM-522.

FIG. 5 is the mass spectral data obtained by HPLC/APCI-MS on AM-524.

FIG. 6 is the mass spectral data obtained by HPLC/APCI-MS on AM-540.

SUMMARY OF THE INVENTION

Phosphocholinate cardenolides having anti-hypertensive activity inmammals and related phosphocholinate compounds have now beenunexpectedly discovered. Analysis of the levels of these compounds inserum can be used to diagnose hypertension in mammals. These compoundscan be obtained and used to treat hypertension in mammals.

In one aspect, the present invention is directed to compounds having theformula ##STR1## wherein R=phosphocholine wherein R₂ =OH or H, if dashedline is absent

wherein the dashed lines represent a double bond which may be present orabsent, and (a) if present in ring B, R₀ is absent; (b) if absent, R₀ isH-β, R₁ is α-H or β-H.

In another aspect, the present invention is directed to compounds havingthe formula ##STR2## wherein R₁ =α-H or β-H; R₂ =H or OH;

R₃ =CH₃, CH₂ --CH₃, CH(CH₃)₂, n-buytl or other alkyl group;

R₄ =H, acetate or other ester; and

R=phosphocholine.

In another aspect the present invention is directed to a method fortreating a mammal suffering from hypertension comprising administeringan effective amount for treating hypertension of a compound having theformula ##STR3## wherein R=phosphocholine wherein R₂ =OH or H, if dashedline is absent

wherein the dashed lines represent a double bond which may be present orabsent, and (a) if present in ring B, R₀ is absent; (b) if absent, R₀ isH-β, R₁ is α-H or β-H.

In yet another aspect the present invention is directed to apharmaceutical formulation for treating hypertension in a mammalcomprising an isolated compound having the structure ##STR4## whereinR=phosphocholine wherein R₂ =OH, if dashed line is absent

wherein the dashed lines represent a double bond which may be present orabsent, and (a) if present in ring B, R₀ is absent; (b) if absent, R₀ isH-β, R₁ is α-H or β-H. and a pharmaceutically acceptable carrier ordiluent.

In a further aspect the present invention is a method for diagnosinghypertension in a patient comprising the steps of:

a) obtaining a serum sample from said patient;

b) determining the amount of a compound selected from the groupconsisting of AM-496, AM-520, AM-522, AM-524 and AM-540;

c) comparing the amount of said compounds determined in step b with theamount of said compounds present in normotensive individuals;

wherein said patient is suffering from hypertension if the amount ofsaid compounds determined from said patient is significantly differentfrom the amount of said compounds present in said normotensiveindividuals.

In another aspect the present invention is directed to a method fordiagnosing pre-menstrual syndrome (PMS) in a female patient comprisingthe steps of:

a. obtaining serum samples from said patient during said patient'smenstrual cycle;

b. determining the levels of AM-496, AM-520, AM-522, AM-524 and AM-540in said samples;

c. comparing the levels of said compounds with those present in serumsamples obtained from normal female patients not suffering from PMS;

d. wherein the levels in any one of said compounds is significantlydifferent in said patient compared to said normal controls, said femalepatient is suffering from PMS.

In another aspect the present invention is directed to a method fortreating a mammal suffering from polycystic kidney disease comprisingadministering to a mammal in need of such treatment a disease treatmenteffective amount of AM-524.

These and other aspects of the present invention will be apparent tothose of ordinary skill in the art in the light of the presentdescription, claims and drawings.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references cited in thisspecification are hereby incorporated by reference in their entirety. Incase of any inconsistences, the present disclosure, includingdefinitions, will prevail.

Cardenolide phosphocholinates having the structure (I) and relatedcompounds having the structure (II) have now been unexpectedlydiscovered. ##STR5## wherein R=phosphocholine wherein R₂ =OH or H, ifdashed line is absent

wherein the dashed lines represent a double bond which may be present orabsent, and (a) if present in ring B, R₀ is absent; (b) if absent, R₀ isH-β, R₁ is α-H or β-H. ##STR6## wherein R₁ =α-H or β-H; R₂ =H or OH;

R₃ =CH₃, CH₂ --CH₃, CH(CH₃)₂, n-buytl or other alkyl group;

R₄ =H, acetate or other ester; and

R=phosphocholine.

The compounds are present in the serum of all mammals tested (human,rat, goat, mouse, pig and cattle). These compounds can be obtained fromnumerous sources, including without limitation, Type 1 human femalebreast cyst fluid (obtained by needle biopsy), bovine adrenal extractsand porcine ovarian follicular extracts. Bovine adrenal glands andporcine ovarian follicles can be obtained from commercial slaughterhouses. The extracts can be prepared as described below. Human breastcyst fluid can be obtained and identified as set forth in U.S. Pat. No.5,028,438 issued Jul. 2, 1991.

Although the phosphocholinate compounds of the present invention arefound in human female breast cyst fluid as a mixture, their structure,interrelationship and physical properties of the individual compounds(indeed, the fact that there are multiple members of the family ofcompounds--see FIG. 1) were not apparent from the disclosure of U.S.Pat. Nos. 5,028,438 and 5,122,371. Phosphocholinate compounds producedpursuant to the present invention may be isolated and purified from themixture disclosed in these patients.

The compounds of the present invention, exemplified by AM-520 or γ,AM-522a or AM-522b or β, AM-524 or α for Structure I and AM-496 or δ forStructure II can be isolated as set forth below. α, β and γ arecardenolides with anti-hypertensive activity in mammals whichcross-react with digoxin-specific antibodies. The structures of α, β, βand δ and their proposed interrelationship is shown in FIG. 1.

When isolated from Type 1 human breast cyst fluid, the compounds may beobtained and purified as set forth in U.S. Pat. No. 5,028,438.

When isolated from tissue (e.g. ovarian follicular tissue or adrenalglands), the tissue is homogenized with a mixture of, by way ofnon-limiting example, methanol, acetone, ethanol and preferablyacetonitrile and water to give a final ratio of between about 10:1 andabout 3:1 and preferably about 4:1. Because of the water present in thetissue, adding a 6:1 ratio of solvents leads to a ratio of 4:1 present.The mixture is filtered using, for example, a Whatman #1 filter (or anyequivalent) and insoluble material is discarded. The volume of thefiltrate is determined and 50% of the volume is added as for exampletoluene, chloroform and preferably benzene to yield a lower aqueous andan upper organic phase. The lower aqueous phase contains most of theactive material but some additional material can be recovered byre-extracting the upper organic phase and combining the aqueous phases.The combined aqueous phases are evaporated to dryness under vacuum,using, by way of non-limiting example, a rotary evaporator withoutallowing the temperature to exceed about 40° C. The evaporated extractis redissolved in a minimum volume (20-50 ml per liter of originalextract) of acetonitrile:methanol:water (4:1:1) and filtered (using, byway of non-limiting example, a Whatman #1 filter) discarding anyinsoluble materials. Alternatively, the lower aqueous phase can befreeze-dried producing a residue. The freezer-dried residue in thisembodiment is dissolved in 20-50 ml per liter of original extract ofacetonitrile:water:methanol (4:1:1). Insoluble material is removed byfiltration as above. In either case, the filtrate is preparativelychromatographed on a RSIL-NH₂ column (Alltech, Chicago, Ill.) (22×500mm/diameter×length) using an acetonitrile:water:methanol gradient withincreasing water concentrations. Alternatively, any amino isopropylcolumn, such as an amino carbohydrate column (Alltech) can be used. Anexample of a suitable gradient is set forth below.

    ______________________________________                                        Time                                                                          (Min)   Acetonitrile                                                                            Methanol   Water Flowrate                                   ______________________________________                                        0       75        20         5     5 ml/min                                   40      65        20         10    5 ml/min                                   40      65        20         10    5 ml/min                                   80      20        20         60    5 ml/min                                   120     20        20         60    5 ml/min                                   130     75        20         5     5 ml/min                                   180     75        20         5     5 ml/min                                   Stop                                                                          ______________________________________                                    

Each fraction is analyzed by HPLC and appropriate fractions identifiedby LC-MS (see below), pooled, lyophilized, redissolved and thepreparative procedure may be repeated as necessary until the LC-MS showsthat only one material is resent. AM-524 and AM-496 have the propertiesset forth below.

    ______________________________________                                        Physical Properties                                                                         AM-524 (α)                                                                         AM-496 (δ)                                     ______________________________________                                        UV absorption maxima                                                                          ˜230 nm                                                                              ˜240 nm                                    Molecular weight                                                                              524 daltons  496 daltons                                      Peaks in mass spectra                                                                         546, 524, 487,                                                                             518, 496, 459,                                   (APCI+)         341, 184     313, 184                                         Acetate derivative                                                                            yes - 628 daltons                                                                          yes- 560 daltons                                 Cross reactivity with                                                                         Yes          No                                               digoxin antibodies                                                            ______________________________________                                    

Assignment of mass fragmentation patterns

    ______________________________________                                                       AM-524 (α)                                                                      AM-496 (δ)                                       ______________________________________                                        Positive ion spectra:                                                         Sodium salt      546       518                                                molecular ion    524       496                                                loss of trimethylamine                                                                         487       459                                                from sodium salt                                                              loss of phosphocholine                                                                         341       313                                                phosphocholine   184       184                                                Negative ion spectra:                                                         loss of methyl   509       481                                                loss of choline  437       409                                                ______________________________________                                    

The above-identified compounds can be assayed by HPLC-Mass spectroscopywith monitoring at the different specific mass ions. The assignments ofthe mass fragmentation patterns of the other compounds of the presentinvention (e.g. AM-520, AM-522 and AM-540) are set forth in Example 1below. In this way, each compound of the present invention can beseparately isolated and identified as each compound elutes in aseparate, distinct fraction on the HPLC gradient. The gradient for theHPLC is 95% acetonitrile: 5% water to 55% acetonitrile: 45% water.Varian (Palo Alto, Calif.) HPLC equipment can be used such as model 9010or 9012 pumps, model 9065 diode array detector (model 9050 singlewavelength detector for preparative HPLC and for the monitor on the massspectrometer), and model 9100 for the autosampler.

The compounds of the present invention can be used in a method fordiagnosing hypertension in a patient. The method comprises obtaining aserum sample (i.e. as little as 0.5 ml) from a patient and determiningthe levels of AM-496, AM-520, AM-522, AM-524 and AM-540 and comparingthe levels with those obtained from normotensive (i.e. non-hypertensive)controls. Indeed, ranges of the levels of the compounds can be obtainedfrom normal individuals so that normal controls need not be obtained foreach assay. If the amount of any of the compounds of the presentinvention is significantly different from those of the control group, apositive diagnosis of hypertension can be made. "significantlydifferent" is defined herein as more than two standard deviations.

Once isolated, the compounds of the present invention can beadministered to patients suffering from hypertension, congestive heartdisease and atrial fibrillation. When administered to patients sufferingfrom hypertension congestive heart disease and atrial fibrillation,AM-524 is preferred because it should be the most active compound ofthis series. AM-520 or AM-522 may also be useful because afteradministration they can be converted in the recipient's body to AM-524.

Without wishing to be bound by theory, it is believed that the compoundsof the present invention will have a better therapeutic index thandigoxin or digitalis. Because these compounds are endogenousanti-hypertensive agents, it is expected that a metabolic degradativepathway exists that regulates the synthesis and breakdown of thesecompounds Therefore, if toxic levels of AM-524 occur, then thedegradation pathway will be stimulated thereby reducing the toxicity. Incontrast, digoxin and related plant compounds do not have a regulateddegradation pathway in mammals and therefore toxic levels canaccumulate.

Large amounts of the compounds of the present invention were found inporcine follicular fluid. This fluid should also be the same as thatpresent in human follicles. In women, each month follicles develop,burst to release the ovum to be fertilized and simultaneously releasethe cardenolides. In one embodiment, the compounds of the presentinvention can be used to diagnose pre-menstral syndrome (PMS) oradministered to women suffering from PMS. Without wishing to be bound bytheory it is believed that in some women, inadequate amounts orinappropriate types of the compounds of the present invention aresynthesized and released. This would cause sodium and water retention ifa Na+-K-ATPase-inhibitory cardenolide of the present inventionpredominates, features that are part of PMS. This could be diagnosed bymeasuring serum levels of the compounds described herein duringdifferent times (i.e. the beginning, middle and end) of a femalesmenstral cycle. At the present time, there is no diagnostic laboratorymeasurement for PMS. If inadequate levels of specific cardenolides wereobserved, then replacement therapy using the compounds of the presentinvention would be used once the specific compounds were identified.This therapy would be expected to treat the cyclic edema that is typicalof PMS. The natural function of these compounds in the ovary may be toregulate the accumulation of electrolytes and water in the developingfollicle. They may also play an important role in the accumulation ofamniotic fluid during pregnancy. Without wishing to be bound by theory,AM-496 is expected to be electrolyte and water retaining similar toaldosterone whereas AM-524 should be electrolyte wasting. Thecardenolides should cause sodium and water loss if AM-524 predominates.

The characteristic feature of the cardenolide compounds of the presentinvention is the presence of the phosphocholine group attached to anaglycone. The presence of the phosphocholine group is confirmed by themass spectral data. The compounds of the present invention are watersoluble, but not soluble in most organic solvents such as ethyl acetate,benzene or isooctane. In the well known Folch solvent mixture (3:4:8,water:methanol:chloroform) these compounds partition into the aqueousphase. The phosphocholinate compounds are all present in high potassiumbreast cyst fluid (Type 1), follicular fluids, adrenal glands and serum,but are not present or are present in much lower concentrations in highsodium breast cyst fluids (Type 2). The materials are not detectable intestes extracts. Extracts from Type 1 breast cyst fluids containingthese compounds were previously shown to cause decreases in bloodpressure when administered to rats. The purification process used wouldhave collected each of the phosphocholinate cardenolides describedherein as a mixture.

AM-540, AM-524, AM-522 and AM-520 all contain cardenolide groups,-similar to the E-ring of ouabain and digoxin, a lactone ring and can beconsidered as digoxin-like or ouabain-like materials. The cardenolidestructures are confirmed by the UV spectra pattern, with absorption atapproximately 220 nm (i.e. 220-240 nm), and cross-reactivity withdigoxin specific antibodies (commercially available from DuPont, Boston,Mass.).

AM-540, AM-524, AM-522, AM-520 and AM-496 are all present in detectablelevels in serum. Measurement of each one will be useful as markers forhypertension, salt accumulation, and in women, cyclic edema or cyclicsalt accumulation. As little as 0.5 ml of serum can be used todetect/identify the compounds of the present invention.

There will be patients with both acquired and congenital deficiencies ofAM-540, AM-524, AM-522, AM-520 and AM-496. The actual compounds will beuseful as replacement therapy for these patients. The phosphocholinatecardenolides will also be useful to treat overdoses of digoxin. Acombined digoxin-phosphocholinate cardenolide mixture described furtherbelow may also be useful to reduce digoxin-related toxicity. AM-524 isparticularly preferred for this use.

It is believed that the compounds of the present invention will alterthe activity of the Na+K+-ATPase enzyme. Cardiac glycosides digoxin anddigitalis inhibit this enzyme activity. Assays for this activity areshown in Example 3 below.

The compounds of the present invention are endogenous hypotensiveagents. For treating mammals afflicted with elevated blood pressureusing the compounds of the present invention, the effective dosage ofthe compounds of this invention depends upon the severity of condition,the stage and the individual characteristics of each mammal beingtreated. For treating hypertension, congestive heart disease or atrialfibrillation in a mammal, it is expected that the compounds of thepresent invention will generally be administered in dosages rangingbetween about 0.01 mg and about 10 mg per kg of body weight per day. Asmentioned above, AM-524 is particularly preferred for this use.

The present invention also provides pharmaceutical formulationscomprising digoxin and the phosphocholinate cardenolides of the presentinvention in a combined dosage form. Such dosage forms would havereduced digoxin-related toxicity. AM-524 is particularly preferred forthis use. The dosage forms would generally contain about 0.05 mg ofdigoxin and about 0.01 mg of AM-524.

The compounds of the present invention may be preferably administeredorally but can also be administered via the parenteral route,intranasally, sublingually, submucosally or by transdermal patch inpharmaceutical compositions prepared by well known methods. Examples ofparenteral dosage forms include aqueous solutions of the compounds ofthe present invention in isotonic saline, 5% glucose or other well knownpharmaceutically acceptable liquids. In a preferred embodiment of thepresent invention, the pharmaceutical compositions can also beformulated so as to be suitable for oral administration. The activeingredient(s) is contained in a capsule (in liquid form) or a tablet.The quantity of effective dose supplied by each capsule or tablet isrelatively unimportant since the total dosage can be reached byadministration of either one or a plurality of capsules or tablets orboth. The capsules employed may comprise any well known pharmaceuticallyacceptable material such as gelatin, cellulose derivatives, etc. Eachcapsule contains an appropriate amount of the compounds of the presentinvention dissolved in a suitable solvent, e.g. ethyl alcohol USP,purified water USP or any conventional aqueous pharmaceutical material.Due to the increased solubility in aqueous media of the compounds of thepresent invention the dosage forms can comprise any aqueouspharmaceutical ingredient.

In an alternate embodiment of the present invention, thephosphocholinate compounds of the present invention can be used to treatpolycystic kidney disease. Without wishing to be bound by theory, it isbelieved that polycystic kidney disease leads to an abnormal buildup ofsome of the intermediate phosphocholinate cardenolide compounds (e.g.AM-520, AM-522, etc.) disclosed herein. It is believed thatadministration of effective amounts of the end-product (AM-524) willlead to a shutdown of the accumulation of the intermediate compounds andan amelioration of the disease. Such effective amounts can be determinedby no more than routine experimentation.

In another embodiment of the present invention preeclampsia can betreated by administering amount of the compounds disclosed hereineffective to treat preeclampsia. Preeclampsia is a toxemia of pregnancycharacterized by the appearance of a constellation of abnormalitieswhich include hypertension, edema and proteinuria. The effective amountswould be the same used when treating hypertension disclosed above.

The invention is further illustrated in specific examples set forthbelow which are intended to describe the invention without limiting itsscope.

Example 1: Proposed Structures of the Compounds of the Present Invention

Proposed structure of AM-496(δ):

R=Phosphocholine

496 daltons--the mass ion

518 daltons--the sodium salt

459 daltons--loss of NMe3 from choline

313 daltons--loss of phosphocholine

Mass spectral data obtained by APCI+ on column fractions. Chromatograms(FIG. 2) show the coincidence of these fragments in the spectra.

The 313 dalton fragment is the aglycone without the 3 hydroxy group andphosphocholine moiety. The 184 dalton fragment is derived from thephosphocholine. The other fragments confirm the breakdown of thephosphocholine.

The structure was further confirmed by the UV spectrum with a peak at240 nm as expected for the D-ring conjugated ketone structure. Theaglycone portion of the molecule has been reported, but not conjugatedto phosphocholine.

When reacted with acetic anhydride in pyridine, a derivative was formedwith a mass of 560 daltons. This corresponds to the monoacetate of thesodium salt. The formation of a single acetate ester under theseconditions confirms the absence of other exposed hydroxy groups andeliminates sugars at the 3 position.

AM-496 has little cross-reactivity with digoxin-specific antibodies.AM-496 is present in serum from all species tested (human, mouse, rat,goat, cattle).

Functionally, this compound should be a weak aldosterone agonist,similar to 21-hydroxyprogesterone (11-deoxycorticosterone, DOC).

Proposed Structures for AM-520(γ):

R=Phosphocholine

520 daltons--the mass ion

542 daltons--the sodium salt

483 daltons--loss of NMe3 from choline

337 daltons--loss of phosphocholine

Mass spectral data obtained by APCI+ on column fractions. Chromatograms(FIG. 3) show the coincidence of these fragments in the spectra.

The 337 dalton fragment is the aglycone without the 3 hydroxy group andphosphocholine moiety. The fragment may be the I-steroid. The peakintensity is relatively small. The 184 dalton fragment is derived fromthe phosphocholine. The other fragments confirm the breakdown of thephosphocholine.

This compound would be formed by dehydration of AM-538. Because of theextended conjugation, there should be a UV peak about 260 nm. A peak atthat wavelength is present, coincidental with the observed mass ions.

The intermediate could be bound to CoA and hydrolysed. The lactone couldform spontaneously, but there may be an enzyme to speed the process.

AM-520 would then be reduced in two steps to produce AM-524.

Proposed structure for AM-522(β):

R=Phosphocholine

522 daltons--the mass ion

544 daltons--the sodium salt

485 daltons--loss of NMe3 from choline

339 daltons--loss of phosphocholine

Mass spectral data obtained by APCI+ on column fractions. Chromatograms(FIG. 4) show the coincidence of these fragments in the spectra.

The 339 dalton fragment is the aglycone without the 3 hydroxy group andphosphocholine moiety. The fragment may be the I-steroid. The peakintensity is relatively small. The 184 dalton fragment is derived fromthe phosphocholine. The other fragments confirm the breakdown of thephosphocholine.

Compound A would be formed by reduction of the A ring and leave intactthe extended conjugation in Ring E. This would lead to a LW peak at 260nm. A peak at that wavelength is present, coincidental with the observedmass ions. The assignment of 5β reduction is based on the correspondingstructures of digoxin and ouabain which are also 5β reduced.

Compound B would be formed by reduction of the D ring. This would leadto elimination of the extended conjugation and loss of the 260 nm peak.Several such compounds were detected in the HPLC chromatographs.

The choice of which compound is reduced first could be determined by therespective enzyme binding characteristics.

Proposed structure for AM-524(α):

R=phosphocholine

524 daltons--the mass ion

546 daltons--the sodium salt

487 daltons--loss of NMe3 from choline

341 daltons--loss of phosphocholine (I-steroid)

Mass spectral data obtained by APCI+ on column fractions. Chromatograms(FIG. 5) show the coincidence of these fragments in the spectra. Furtherconfirmed by UV spectra with peak at 220 nm, which is characteristic ofthe cardenolide structure. Note the absence of the 14 hydroxy group andthe tentative assignment of 14α hydrogen on the bridge. This assignmentchosen to be the same as that of other steroid hormones. Also note theassignment of the 5β hydrogen on the basis of the similar structure inthe other members of the cardenolide series. The 341 dalton peakcorresponds to loss of the phosphocholine and could be the I-steroidderivative.

When reacted with acetic anhydride in pyridine, a derivative is formedwith a mass ion at 628 daltons. Acetic anhydride would react with thelactone to form an ester and a free carboxylic acid. The compound withthe carboxylate anion would not be detected in the cation spectra. To bedetected as a cation, the carboxylate would have to be the sodium salt.The combination of the ester and the sodium salt would add 82 daltons tothe mass, exactly as observed. This derivative indicates that there canbe no other free hydroxyl groups in the molecule, for example, no sugarscan be coupled at the three position of the aglycone.

The phosphocholine is confirmed by the fragmentation pattern. Thepresence of a 184 dalton fragment is characteristic, along with thefragments corresponding to loss of trimethylamine from the sodium salt.In the negative ion spectra, the largest mass corresponds (509 daltons)to loss of methyl and the base ion derives from complete loss of choline(loss of 87 dalton fragment--leaving a major fragment at 437 daltons).AM-524 cross-reacts with digoxin specific antibodies. AM-524 is presentin serum from all species tested (human, mouse, rat, goat, cattle).

Proposed structures for AM-540:

R=Phosphocholine

540 daltons--the mass ion

503 daltons--loss of NMe3 from choline

357 daltons--loss of phosphocholine (I-steroid)

Mass spectral data obtained by APCI+ on column fractions. Chromatograms(FIG. 6) show the coincidence of these fragments in the spectra.

The 357 dalton fragment would be the aglycone without the 3 hydroxygroup and phosphocholine moiety, but it was not observed. The 184 daltonfragment is derived from the phosphocholine. The other fragments confirmthe breakdown of the phosphocholine.

Both structures shown should generate the observed fragmentationpatterns. However, structure A would be a precursor for AM524 whilestructure B would be a further metabolite, perhaps most like digoxin.The B structure requires the activity of a 14 hydroxylase which has notbeen reported in mammalian systems. The two possibilities could beresolved by examination of the UV spectrum of the pure material becauseB should have a UV peak at 220 nm but A should not. The aglycone portionof B has been reported, but not conjugated to phosphocholine.

The hydroxy group is not limited to the 14 position. The observedmaterial could be a metabolite on the degradative pathway, rather thanthe most active cardiotonic compound.

Example 2: Assay for Anti-hypertensive Activity

Sprague-Dawaley rats, weighing 270-300 grams, will be used in allexperiments. Rats will be anesthetized with a single intraperitoneal(ip) injection of pentobarbital, 30-40 mg/kg body weight. Polyethylenecatheters will be injected into the carotid artery, left externaljugular vein and bladder. Upon completion of the surgical procedure, theanimals will be given a priming injection of 0.15 M NaCl solution equalto 0.5% body weight. In order to monitor renal function, an infusion of0.15 M NaCl solution will be started at 0.045 ml/min and containing ¹⁴C-inulin and ³ H-PAH (para-aminohippuric acid, New England Nuclear,Boston, Mass.) in quantities designed to provide 5 microCi per hour and10 microCi per hour of the 2 radioactive isotopes, respectively. Afteran equilibration period of 40-50 minutes, the experiment will becommenced.

The intra-arterial blood pressure will be recorded using a transducerand a 8-channel physiography (Beckman, Palo Alto, Calif.). The pressuretracing will be calibrated using a manual sphygmomanometer. Hematocritvalues will be measured by routine techniques well known in the art. Theplasma and urinary activity of ¹⁴ C-inulin-³ H-PAH will be measuredusing a beta scintillation counter (Packard Tricarb, PackardInstruments, Chicago, Ill.) The plasma electrolytes will be measuredusing a flame photometer (Klinaflamer, Beckman Instruments, Palo Alto,Calif.). The glomerular filtration rate (GFR) and renal blood flow willbe calculated from the 14C-inulin and ³ H-PAH clearances, respectively,using standard equations.

AM-524, AM-540, AM-496 and a digoxin control will be administeredintravenously at concentrations ranging between 0.01 μg/kg and 10 μg/kgbody weight. Blood pressure will be recorded continuously for theduration of the study.

Example 3: Reactivity of the Phosphocholinate Cardenolide Compounds ofthe Present Invention with Digoxin-Specific Antibodies

A typical assay will be performed as follows. Standard solutions,containing known amounts of digoxin, will be prepared by dilutingstandards provided by the manufacturer (Dupont, Boston). Standard curveswill be prepared by adding 0.1 ml of standard solutions containing 0.05,0.1, 0.2, 0.5, 1.0 and 2.0 ng/ml of digoxin to 12×75 mm borosilicateculture tubes (Fisher Scientific Co.) and assaying as described below.

Samples of AM-524, AM-540 and AM-495 are pipetted into 12×75 mm culturetubes and evaporated to dryness under a stream of nitrogen. 0.2 ml of[¹²⁵ I]-labeled digoxin-tracer and 0.2 ml of digoxin antibody are addedto each tube. The tubes are mixed by vortex and allowed to incubate for30 minutes at room temperature. Thereafter, the tubes are centrifuged at2600xg at 4° C. for 30 minutes and transferred to foam decanting racks(Diagnostic Products, Los Angeles, Calif.) and decanted. Each tube willbe blotted and the amount of [¹²⁵ I] is determined using a gamma counter(Model 1275 Minigamma, LKB, Gaithersburg, Md.). Individual values ofdigoxin are determined by interpolation against the standard curveobtained at the same time using a smooth line best fit program from LKBand are expressed as ng digoxin equivalents, as described above.

Example 4: Distribution of Phosphocholinate Cardenolides Obtained FromNormal Women and Women Suffering From PMS

Serum samples will be obtained from women suffering from PMS andunaffected control women at different times in their menstral cycle(i.e. before, during and after). The serum samples will be processed asdescribed above and analyzed by LC-MS for the presence of the compoundsof the present invention. Any difference in the levels of any one of thecompounds of the present invention greater than two standard deviationscan be considered a positive diagnosis of PMS. It is expected that womensuffering from PMS will have cyclic fluctuations in their serum levelsof the compounds of the present invention, mirroring the episodes oftheir symptoms.

Example 5: Distribution of Serum Phosphocholinate Cardenolides ObtainedFrom Normal Individuals and Hypertensive Patients and their Families

Genes related to the synthetic pathway for the phosphocholineatecompounds of the present invention can be identified by determining theratios of AM-496, AM-520, AM-522 and AM-524 and AM-540 in individualsand their family members. Serum samples will be obtained from normalindividuals and those suffering from hypertension, hypotension, renaldisorders (e.g. nephritis or polycystic disease) salt-wasting disorders(e.g. hyperaldosteronism, hydroxylase deficiency, Addison's disorder)(hereinafter alternatively referred to as the proband) and their familymembers. The samples will be extracted with acetonitrile and subjectedto LC-MS as described above. Ratios of AM-496 to AM-524, AM-520, AM-522,and AM-540 will be determined. If the proband had normal ratios thefamily would not be further investigated or can be used as a controlfamily. If the proband had abnormal ratios, then the family members willbe investigated. If the disorder of the proband was also present in someof the family members, then the relationship between the two events willalso be determined. If the disorder itself cannot be identified infamily members, the abnormal ratio pattern might be present. This couldbe used for risk assessment as well as for genetic studies.

What is claimed is:
 1. An isolated, purified compound having the formula##STR7## wherein R=phosphocholine wherein R₂ =OH or H, if dashed line isabsentwherein the dashed lines represent a double bond which may bepresent or absent, and (a) if present in ring B, R₀ is absent; (b) ifabsent, R₀ is H-β, R₁ is α-H or β-H.
 2. The compound of claim 1 whereinsaid compound is selected from the group consisting of AM-520, AM-522,AM-524 and AM-540.
 3. A method for treating a mammal suffering fromhypertension comprising administering an effective amount for treatinghypertension of a compound having the formula ##STR8## whereinR=phosphocholine wherein R₂ =OH or H, if dashed line is absentwhereinthe dashed lines represent a double bond which may be present or absent,and (a) if present in ring B. R₀ is absent; (b) if absent, R₀ isH-β_(n), R₁ is α-H or β-H.
 4. The method of claim 3 wherein saidcompound is selected from the group consisting of AM-520, AM-522, AM-524and AM-540.
 5. The method of claim 4 wherein said compound is AM-524. 6.A pharmaceutical formulation for treating hypertension in a mammalcomprising an isolated compound having the structure ##STR9## whereinR=phosphocholine wherein R₂ =OH, if dashed line is absentwherein thedashed lines represent a double bond which may be present or absent, and(a) if present in ring B. R₀ is absent; (b) if absent, R₀ is H-β_(n), R₁is α-H or β-H. and a pharmaceutically acceptable carrier or diluent. 7.A method for diagnosing hypertension in a patient comprising the stepsof:a) obtaining a serum sample from said patient; b) determining theamount of a compound selected from the group consisting of AM-496,AM-520, AM-522, AM-524 and AM-540; c) comparing the amount of saidcompounds determined in step b with the amount of said compounds presentin normotensive individuals; wherein said patient is suffering fromhypertension if the amount of said compounds determined from saidpatient is significantly different from the amount of said compoundspresent in said normotensive individuals.
 8. A method for diagnosingpre-menstrual syndrome (PMS) in a female patient comprising the stepsof:a) obtaining serum samples from said patient during said patient'smenstrual cycle; b) determining the levels of AM-496, AM-520, AM-522,AM-524 and AM-540 in said samples; c) comparing the levels of saidcompounds with those present in serum samples obtained from normalfemale patients not suffering from PMS; d) wherein the levels in any oneof said compounds is significantly different in said patient compared tosaid normal controls, said female patient is suffering from PMS.
 9. Themethod of claim 8 wherein said significant difference is greater thantwo standard deviations when compared to normotensive controls.
 10. Amethod for treating a patient suffering from PMS comprisingadministering an effective amount for treating PMS of a compound havingthe formula ##STR10## wherein R=phosphocholine wherein R₂ =OH or H, ifdashed line is absentwherein the dashed lines represent a double bondwhich may be present or absent, and (a) if present in ring B, R₀ isabsent; (b) if absent, R₀ is H-β, R₁ is α-H or β-H.
 11. A method fortreating a mammal suffering from preeclampsia comprising an effectiveamount for treating pre-eclampsia of a compound having the formula##STR11## wherein R=phosphocholine wherein R₂ =OH or H, if dashed lineis absentwherein the dashed lines represent a double bond which may bepresent or absent, and (a) if present in ring B, R₀ is absent; (b) ifabsent, R₀ is H-β, R₁ is α-H or β-H.
 12. A method for diagnosingpre-eclampsia in a female patient comprising the steps:a) obtainingserum samples from said patient during said patient's menstrual cycle;b) determining the levels of AM-496, AM-520, AM-522, AM-524, and AM-540in said samples; c) comparing the levels of said compounds with thosepresent in serum samples obtained from normal female patients notsuffering from pre-eclampsia; d) wherein if the levels in any one ofsaid compounds is significantly different in said patient compared tosaid normal female patients not suffering from pre-eclampsia, saidfemale is suffering from pre-eclampsia.
 13. The method of claim 12wherein said significant difference is greater than two standarddeviations when compared to unaffected control women.
 14. An isolated,purified compound having the formula: ##STR12##
 15. A method fortreating a mammal suffering from polycystic kidney disease comprisingadministering to a mammal in need of such treatment a disease treatmenteffective amount of a compound having the formula: whereinR=phosphocholinewherein R₂ =OH or H, if dashed line is absent whereinthe dashed lines represent a double bond which may be present or absent,and (a) if present in ring B, R₀ is absent; (b) if absent, R₀ is H-β, R₁is α-H or β-H.
 16. A pharmaceutical formulation comprisinga) digoxin, b)a compound having the formula ##STR13## wherein R=phosphocholine whereinR₂ =OH or H, if dashed line is absent wherein the dashed lines representa double bond which may be present or absent, and(i) if present in ringB, R₀ is absent; (ii) if absent, R₀ is H-β, R₁ is α-H or β-H; and c) apharmaceutically acceptable carrier or diluent.